The concept of immune system activation in the setting of thoracic malignancy dates to the 1970’s, when investigators noted improved survival in patients with empyema after resections for lung cancer [4], justifying largely unsuccessful trials of intrapleural Baccilus Calmette-Guérin (BCG) [5] and other bacterial antigens. Direct instillation of the recombinant cytokines interferon ɣ [6], interferon α2b [7] and IL-2 [8, 9] have also been tested. Intrapleural IL-2 was well tolerated in non-small cell lung cancer (NSCLC). Further, intrapleurally administered IL-2 levels were 6000-fold higher than in the plasma [8], indicating that locally administered IL-2 (formula weight = 15.5 kDa) is sequestered in the pleural space. This is a very important observation, since other large molecule biologicals, such as antibodies can be expected to be similarly concentrated when administered directly to the pleura. Although intrapleural IL-2 cleared effusions in 28 of 31 patients studied [9], and partial responses were seen with other cytokine modalities, the median time to progression ranged from days [7] to months [8]. Thus instillation of high-dose Th1 associated cytokines was insufficient by themselves to overcome the immunosuppressive environment of the pleural space.

A recent clinical trial described the use of tumor infiltrating lymphocytes (TIL) derived from MPE and malignant ascites in combination with cisplatin [10]. Effusion-derived TIL provided longer progression-free survival and better quality of life than cisplatin alone. The combination of immunotherapy and chemotherapy is somewhat counterintuitive since many cytotoxic agents inhibit cell proliferation, an important aspect of adaptive immunity. However, regulatory T cells (Treg) have been shown to be more susceptible to platinum-based combination chemotherapy than conventional CD4+ T cells [11], potentially providing a release from Treg-mediated suppression of anti-tumor immunity. Effusion-derived TIL have several important advantages over TIL derived from primary tumors or biopsies of solid metastases: [1] The T-cell yield from MPE or ascites is orders of magnitude higher than from biopsies, so the number of passages required is lower, and culture time is shorter; [2] Malignant effusion pleural T cells (PIT) represent a cross-section of all TIL, whereas TIL derived from solid tumors display spatial heterogeneity and can differ with respect to function and specificity depending on the biopsy location [12]. Owing to their abundance, it may be possible to prime PIT by short term ex vivo exposure to activation signals, and reinstill them without expansion. The resulting cells would not be cytokine addicted as are conventional TIL and would not require administration of high dose IL-2 for their survival.

PD-L1 is expressed on malignant mesothelioma [13] and other malignancies and is therefore potentially targetable by anti-PD-L1 antibodies. T cells from NSCLC MPE display increased expression of PD-1, TIM-3, and CTLA-4, when compared with non-malignant controls [14], possibly due to high levels of TGF-β in the effusion, secreted by PD-L1+ tumor-associated M2-macrophages.

It is becoming increasingly clear that both conventional and immunotherapeutic attempts have failed in MPE because the pleural space is a sequestered environment in which tumor cells and immune cells interact to the benefit of the tumor. In the pleural space, wound-healing cytokines and chemokines are concentrated, and juxtacrine interactions of tumor, macrophages and mesothelial cells are favored by their proximity. The result is the perpetuation of a wound-healing environment in which T-cell effectors are suppressed or killed, and macrophages are channeled to an M2 program that assists angiogenesis and metastasis, all culminating in the promotion of an aggressive and invasive EMT tumor phenotype.

The pleural space represents a sequestered local environment formed by mesothelial cells joined by tight junctions [15]. Protein biologics such as IL-2 remain highly concentrated when administered intrapleurally, with local concentrations thousands of times higher than that of the plasma [8]. Protein movement from the plasma to the pleura is also impeded, albeit to a lesser extent, and the pleural effusion to plasma ratio of protein concentrations is inversely related to their molecular weight [16]. This is highly relevant to systemic administration of antibody therapeutics, which predictably would not pass easily into the pleural space, abdominal cavity, or interstitial spaces [17].

The cell-free serous component of MPE contains an array of cytokines and chemokines [18]. The majority of the secreted cytokines in MPE are Th2-like and include IL-10 [19], VEGF [20] and TGFβ [21], further promoting the wound-healing milieu to the detriment of an anti-tumor effector response. Interestingly, the pleiotropic cytokine IL-6 and its soluble receptor component sIL-6Rα are among the most abundant cytokines in MPE [22]. IL-6 is produced by the tumor [23] and also by pleural mesothelial cells [24] and stromal cells [25]. IL-6 plus IL-10 upregulate PD-L1 expression in tumor cells [26]. IL-6 signal transduction is mediated through a receptor complex consisting of IL-6Rα (CD126) and IL-6Rβ (CD130). IL-6Rβ is expressed ubiquitously, but IL-6Rα expression is restricted chiefly to leukocytes and hepatocytes. In normal physiology, IL-6 mediates powerful systemic effects by trans-signaling, which occurs when IL-6 binds to soluble IL-6Rα and complexes with membrane-bound IL-6Rβ [27]. IL-6 trans-signaling has been shown to promote aggressive tumor behavior and progression in malignant ascites of ovarian cancer [28] and in breast cancer pleural effusions [23], and promotes EMT in non-small cell lung cancer [29], making it an attractive therapeutic target. Tocilizumab, a monoclonal antibody directed against IL-6Rα, is licensed for the treatment of rheumatoid arthritis, and has been used experimentally to treat cancer-associated cachexia [30] and cytokine release syndrome [31]. Intrapleural administration may have profound effects on the polarization of the pleural immune environment with minimal systemic effects.

The proximity and high concentration of T cells, macrophages, mesothelial cells and tumor in the pleural space favors cell-cell contact and juxtacrine signaling. Examples include the promotion of EMT by binding of CD90 and EphA4 on the tumor to CD11b and Ephrin on macrophages, respectively [23]. Similarly, PD-L1 and PD-L2 expressed on tumor and pleural macrophages bind to PD-1 on T cells, promoting anergy, development of induced regulatory T cells (iTregs) and apoptosis [32]. Other ligands expressed on pleural tumor, such as CEACAM1 which binds to TIM-3, may interact with immune checkpoint receptors expressed on PIT. MPE, which are routinely therapeutically drained, provide a unique window into interactions that are more difficult to observe in other metastatic settings.